PROJECT SUMMARY/ABSTRACT Understanding how cell fate is maintained or altered during normal development or disease is of fundamental importance for biomedical research. Mitosis is a period of particular stress on cell identity, as transcription is halted, transcription factors (TFs) leave the chromatin, and chromatin collapses into a cell type invariant structure. This poses a challenge for all cycling cells, but in particular for pluripotent stem cells (PSCs), which proliferate rapidly and must retain the capacity to both self-renew and differentiate into any somatic cell type. Recent studies reported a partial persistence of selected epigenetic marks and TFs on mitotic chromatin, a phenomenon known as mitotic bookmarking, and provided evidence for its importance in stem cell identity maintenance. However, the role and underlying mechanisms of mitotic bookmarking in the propagation of PSC identity remain elusive. Furthermore, little is known about the extent or nature of molecular destabilization during mitosis in PSCs, as most existing information is from somatic cells. The long-term goal of this project is therefore to elucidate mechanisms by which PSCs maintain or change their cell identity. The specific objective is to determine the functional role of mitotic bookmarking in the inheritance of the stem cell identity after mitosis. Genome-wide assays assessing transcription and chromatin architecture will be used to characterize the dynamic molecular reestablishment of PSC identity upon mitotic exit and to determine the extent to which mitotic bookmarking is predictive of faster resetting (Aim 1). These findings will be functionally validated by utilizing tools to degrade bookmarking TFs specifically during mitotic exit and then by assessing the consequent changes in molecular resetting and maintenance of pluripotency (Aim 2). The central hypothesis is that, in PSCs, bookmarking factors act to prime bookmarked regions to be reset more quickly and faithfully than non-bookmarked regions, and that the rapid reactivation of these genes (largely stem cell-specific genes) is necessary for the maintenance of stem cell identity. The expected outcome of this project is to reveal the significance of mitotic bookmarking in stem cell identity and cell fate determination. Successful completion of the aims will provide the knowledge and impetus to utilize the period of destabilized stem cell identity during mitosis to efficiently generate cell types of biomedical relevance. It will also provide mechanistic insight into a potential cell-type specific degree, necessity, and nature of bookmarking. Furthermore, given that dysregulation of cell identity is responsible for various diseases and malignancies, insights into the driving forces that direct these aberrant cell identity changes can help to identify new drug targets and therefore possible treatment options.